ABSTRACT Project #3: Ubiquitin E3 ligases in SSc Systemic sclerosis (SSc) is a progressive multi-organ system fibrotic disorder associated with autoimmunity. The leading causes of SSc-related death are pulmonary hypertension (PAH) and interstitial lung disease (ILD). PAH affects 10-15% of SSc patients. The prevalence of ILD varies from 40 to 65% in cohort studies. SSc-ILD is the subject of CORT Project #3. Common to many of the SSc disease phenotypes, include the skin and the lung, is the observation that fibroblasts, the principal effector cells of fibrosis, assume the contractile myofibroblast phenotype, synthesize matrix, and exhibit a pathologically increased lifespan. A traditional approach to discover novel genes involved in the pathogenesis of pulmonary fibrosis has been to perform genome-wide screening to uncover dysregulated RNA species that may be involved in the pathogenesis of disease. However, this approach may miss the critical role of proteins that are NOT regulated at the level of transcription but rather through protein degradation, particularly through the ubiquitin-proteasome system (UPS) mediated by ubiquitin E3 enzymes. Our recent studies using a large number of idiopathic pulmonary fibrosis (IPF) and SSc lung samples have uncovered an array of several, previously unsuspected, molecular targets. In particular, we have identified two highly novel pro- fibrotic signaling pathways in SSc fibroblasts: First is the loss of a new collagen 1 gene repressor, E2 transcriptional factor 8 (E2F8) by a ubiquitin E3 ligase, Fbxo16. This ultimately leads to increased collagen synthesis in SSc fibroblasts. The second is a new protein isoform, termed FIEL1 (Fibrosis- Inducing E3 Ligase 1), which potently stimulates the central pro-fibrotic cytokine, TGF?, signaling pathway through the site-specific ubiquitination and degradation of the TGF? inhibitor PIAS4. Further, we have developed a first-in-class small molecule inhibitor towards FIEL1 that is highly effective in ameliorating fibrosis in mice. Thus, we hypothesize that dysregulation of members of E3 ubiquitin ligase system drives the fibrotic phenotype in SSc. In this project, we propose to screen SSc-ILD lungs and lung fibroblasts derived from SSc-ILD lungs for dysregulated expression of members of the SCF- and HECT-domain ubiquitin E3 ligases and to assay their function in mediating the pathologic myofibroblast phenotype. We will then design small molecule inhibitors for these E3 ligases and test them for effectiveness in animal models of lung fibrosis as well as our unique ex vivo diseased human lung perfusion and culture systems here at the University of Pittsburgh. These proposed studies will help us discover a new set of potentially druggable targets underlying the pathobiology of fibrotic pathways in SSc. These studies will provide a fundamental platform for a unique and potentially transformative initiative in SSc-ILD. Our project will interact very closely with the other CORT projects and cores to discover the overlapping biology of dysregulated ubiquitin E3 ligases in SSc-PAH and SSc-skin disease.